Aggressive cancer cells often upregulate GRP78, a major endoplasmic reticulum (ER) chaperone and key regulator of the unfolded protein response (UPR) to augment protein folding capacity and maintain ER homeostasis, thereby promoting survival and acquiring therapeutic resistance. A high-throughput drug screen revealed that a class of compounds referred to as cardiac glycosides (CGs) such as digoxin (DIG), in current use to treat heart failure, as novel inhibitors of GRP78 stress induction in a wide range of human cancer. The discovery that CGs can suppress stress induction of GRP78, which is pivotal for cancer survival, invasion and oncogenic signaling, opens up a new mechanism for the antineoplastic action of the CGs. Among the CGs, oleandrin (OLN) with 100-fold higher affinity for the ?3 versus the ?1 subunit of Na-K ATPase and high tolerability, is most potent compared to DIG and other CGs. Here we propose to test the efficacy of OLN to suppress GRP78 and cell viability, taking advantage of unique sets of preclinical patient-derived breast and colon cancer models that closely recapitulate the in vivo milieu developed by our collaborative team. We hypothesize that OLN, acting through the ?3 subunit of the Na-K ATPase, acutely suppresses stress induction of both intracellular and the cell surface form of GRP78 through blocking its acute translation following ER stress. OLN may also preferentially suppress translation of other proteins critical for survival under stress. As a consequence, ER homeostasis is perturbed and ER-stress mediated apoptosis is triggered. These contribute majorly to the antineoplastic mechanism of OLN. In this proposal, we will: 1) establish the efficacy of OLN and for comparison, DIG, in suppressing GRP78 expression, cell viability, stemness and invasion in patient-derived breast circulating tumor cells both in vitro and in vivo; 2) establish the efficacy of OLN and DIG in lowering GRP78 expression and viability of patient-derived colorectal cancer cells and enhancing standard therapy in organoids. Promising treatment schedules will be validated in PDX models in vivo. The GRP78 expression levels, the status of the ?3 subunit of Na-K ATPase and the OLN/DIG concentration in the plasma and tumor in the model systems will be monitored; and 3) examine the mechanisms whereby OLN and DIG suppress stress induction of GRP78 and its impact on the UPR and apoptosis as well as the importance of GRP78 in the anti-cancer effect of OLN/CG. In summary, our studies will provide critical preclinical evaluation of the efficacy of OLN/DIG in cancer leveraging novel patient-derived breast and colon cancer model systems, and a high content imaging platform that mimics the hypoxic tumor microenvironment for monitoring the response. Our studies will also explore the utility of GRP78 and ?3 subunit of Na-K ATPase as biomarkers for stratifying patients for OLN/DIG therapy and drug response, and provide proof-of-concept for further development of CG agents in combination or targeted therapy settings in suppressing GRP78, impacting the UPR and apoptosis. Our results will have far-reaching impact as they are also applicable to other solid and blood cancers dependent on GRP78 for growth, invasion and resistance.